CONTRIBUTION OF INTERSPECIFIC HYBRIDIZATION TO SUNFLOWER BREEDING

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1 HELIA, 35, Nr. 57, p.p , (2012) UDC : : DOI: /HEL C CONTRIBUTION OF INTERSPECIFIC HYBRIDIZATION TO SUNFLOWER BREEDING Christov, M. * Dobroudja Agricultural Institute, 9520 General Toshevo, Bulgaria Received: November 02, 2012 Accepted: December 10, 2012 SUMMARY This investigation is directed at improving sunflower using hybrid forms resulted from interspecific hybridization. The aim is to create new B/A and R lines from interspecific hybrid forms that are resistant to diseases, the parasite broomrape, herbicides, and other stress factors and are characterized with high combining ability and to obtain on this basis highly productive oilseed sunflower hybrids with varied fatty acid composition of oil. The investigation was carried out during the period It involved 16 cultivars and 18 B lines with their analogues as well hybrid material originated from 38 Helianthus species - 9 annuals and 29 perennials. Intraspecific hybridization and purposeful selection were the methods used. Crossing between interspecific hybrids and crossing of interspecific hybrids with sunflower cultivars or lines were applied. Self-pollination, sib-pollination, backcross with pollen from cultivated sunflower and pollination with pollen from different interspecific hybrids were carried out. Phytopathological and biochemical evaluation of seeds and plants and morphological characterization of sunflower forms, lines and hybrids were performed. New sunflower forms and lines were created as a result of this investigation. A greater part of them possess resistance to downy mildew, phomopsis, phoma and alternaria, tolerance to sclerotinia and full resistance to the different races of the parasite broomrape. The new forms were distinguished by new plant architecture, different vegetation period and seeds with different size and coloration. New B/A and R lines having high combining ability and seed oil and fatty acid contents were obtained. Fifteen sources of cytoplasmic male sterility (cms) were obtained from interspecific hybrid forms, as were 251 sources of genes for restoration of fertility (Rf genes). Five new hybrid cultivars of the oilseed type that were obtained on the basis of these lines were created and registered. The obtained results from the investigations showed that by interspecific hybridization new genetic material was transferred to the cultivated sunflower. These results have augmented the contribution of interspecific hybridization to sunflower breeding. Key words: Helianthus, hybridization, hybrids, lines, sunflower * Corresponding author: misho_h5@abv.bg

2 38 HELIA, 35, Nr. 57, p.p , (2012) INTRODUCTION The implementation of interspecific hybridization for practical purposes began at the start of the 20 th century with Saciperov s attempts (1916). These and other later investigations showed that as a result of crossing different Helianthus species with cultivated sunflower new sunflower forms resistant to different diseases and the parasite broomrape could be obtained (Pustovoit V., 1960; Pustovoit G., 1975; Put and Sackston, 1957 and 1963; Leclercq et al., 1970; Fick et al., 1974; Škorić, 1985; Jan and Chandler, 1985; Christov, 1990; Christov, 1996; Christov et al., 1996; Hristova-Cherbadji, 2007; Christov, 2008; etc). Leclercq (1969) found the first cms source originated from the cross between H. petiolaris Nutt. and cultivated sunflower. Other cms sources were found by Whelan (1980), Vranceanu et al. (1986), Serieys and Vincourt (1987), Christov (1990a), Christov (1999), and others. Soon after the discovery of the first cms source, the sources of Rf genes were also found by Kinman (1970) and Enns et al. (1970). The discoveries of Leclercq (1971), Fick et al. (1974), Christov and Petrov (1988), and others followed in this direction. Investigations of Helianthus species were carried out in some other directions. In this report we present the results of investigation of interspecific sunflower hybrids and the use of the hybrid material obtained for developing lines with economically important characters suitable for use as parental components of new sunflower hybrid cultivars. MATERIAL AND METHODS The investigations were carried out at the Dobroudja Agricultural Institute, General Toshevo during the period Plant material In the investigation we included hybrid material originated from 38 Helianthus species and 16 cultivars and 18 lines and their sterile analogues of cultivated sunflower Helianthus annuus. Methods used Methods of intraspecific hybridization and selection were used. They were in the form of crossing between interspecific hybrids and crossing of interspecific hybrids with sunflower cultivars and lines. Self-pollination, sib-pollination, backcross with pollen from cultivated sunflower and pollination with pollen from different interspecific hybrids were implemented. Evaluation of resistance to diseases and broomrape of the studied plant material was carried out using methods confirmed at the Institute (Panchenko, 1975; Tourvieille et al., 1988; Christov, 1990; Christov et al., 1992; Christov, 1996;

3 HELIA, 35, Nr. 57, p.p , (2012) 39 Christov et al., 1996; Fernandez-Martinez et al., 2000; Encheva and Kiryakov, 2002; Christov et al., 2004; Shindrova, 2006). Seed oil and protein contents and fatty and amino acid contents were evaluated according to methods confirmed at the Institute (Rushkovskiy, 1957; Stoianova and Ivanov, 1968; Ivanov et al., 1996). A Nuclear Magnetic Resonance apparatus and the analyzer Hitachi L-8500 were used for the evaluation of seed oil content and for amino acids, respectively. Morphological characteristics were assessed based on phenological observations and biometric measurements during the vegetation period and on the basis of laboratory studies of whole plants and of seed material obtained from them. Sources of cms were sought among materials obtained from crosses wild species cultivated sunflower. For obtaining sterile inflorescences pollination was carried out with pollen from B lines or cultivars. Firstly, sources of Rf genes were sought in crosses sterile sunflower lines wild species. The presence of Rf genes in the genome of wild species was established in F 1. Forms with Rf genes were also found in materials obtained from crosses cultivated sunflower (B line or cultivar) wild species and wild species cultivated sunflower. The development of sunflower B lines was carried out by purposeful selection in the hybrid materials, which in most cases began after the third generation. Evaluation and selection of materials began on the basis of their morphological, biochemical and phytopathological characteristics, the absence of Rf genes, and the presence of good combining abilities. The development of sterile analogues - A lines began with the establishment of the fact that there were not any Rf genes in the studied material. After the BC 3 or BC 4, the study of the general combining ability of the A lines developed began, and after that their specific combining ability was analyzed. The development of self-pollinated lines, the restorers of fertility - R lines, was carried out mainly from crosses of male sterile lines with different wild Helianthus species. Purposeful and repeated selection and self-pollination of fertile plants was implemented until homozygous Rf genes were obtained. The obtained R lines, which possessed 100% fertility restoration and other important characters, were collected from both parental forms included in the hybridization. For creating R lines hybrids obtained from crosses cultivated sunflower wild species and wild species cultivated sunflower could be used. Origin of interspecific hybrids RESULTS AND DISCUSSION As a result of hybridization between sunflower Helianthus annuus and 38 species from the genus Helianthus a total of 67,000 F 1 hybrid plants were obtained

4 40 HELIA, 35, Nr. 57, p.p , (2012) from all the species included in the investigation (Table 1). Except for F 1 hybrids originated from H. simulans all the other F 1 hybrids gave seeds. Table 1: Species of genus Helianthus used in hybridization Groups of species Species Annual species (2n=34) Perennial diploid species (2n=34) Perennial tetraploid species (2n=68) Perennial hexaploid species (2n=102) H. argophyllus, H. bolanderi, H. debilis, H. exilis, H. neglectus, H. paradoxus, H. petiolaris, H. praecox, H. annuus (w.f.)** H. divaricatus, H. doronicoides*, H. giganteus, H. smithii, H. glaucophyllus, H. grosseserratus, H. maximiliani, H. microcephallus, H. mollis, H. nuttallii, H. occidentalis, H. orgialis*, H. pumilus, H. salicifolius, H. silphioides, H. simulans H. decapetalus, H. hirsutus, H. laevigatus, H. scaberimus*, H. tomentosus* H. eggertii, H. pauciflorus (rigidus), H. strumosus, H. resinosus, H. tuberosus, H. ciliaris, H. xlaetiflorus, H. californicus * Not included in classification of Shilling and Heiser (1981); ** Wild form Creation of new sunflower forms from interspecific hybrids The main reason for including wild Helianthus species in the research work on biological improvement of sunflower crop was the presence of resistance to diseases, parasites and pests. Studies on downy mildew resistance were the priority, and they were followed by studies on resistance to sclerotinia, phomopsis, phoma, alternaria, etc. Great priority was given to studies on broomrape resistance. New sunflower forms resistant / tolerant to diseases and parasites Among the hybrid forms with resistance to diseases and broomrape, the highest percentage were those with resistance to the pathogen Plasmopora helianthi. Full resistance to Plasmopora helianthi race N o 700 was observed in more than 2,600 accessions, obtained with participation of 36 Helianthus species (Table 1). Resistance to race N o 731, considered as the most virulent in Bulgaria, was established in more than 400 hybrid forms, originated from the species H. divaricatus, H. hirsutus, H. pauciflorus (rigidus), H. debilis, H. paradoxus, etc. Some of these forms possessed resistance to some other diseases and to the parasite broomrape (Table 2). Table 2: Characterization of sunflower lines, obtained by interspecific hybridization, resistant to downy mildew - race 731, harvest 2009 Accession, pedigree Resistance to Seed oil downy mildew, % broomrape, % content, % Generation PR-1/8 (c.s. H. pauciflorus) PR-9/8 (c.s. H. tuberosus) PR-13/8 (c.s. H. pumilus) PR-25/8 (c.s. H. pauciflorus) PR-35/8 (c.s. H. hirsutus) PR-41/8 (c.s. H. divaricatus)

5 HELIA, 35, Nr. 57, p.p , (2012) 41 Resistance / tolerance to the pathogen Phomopsis helianthi was exhibited by more than 80 forms. They originated from the species H. annuus (w.f.), H. argophyllus, H. debilis, H. glaucophyllus, H. laevigatus, H. eggertii and H. pauciflorus. High resistance to Phoma helianthi was established in several forms, obtained with participation of the species H. eggertii, H. laevigatus, H. argophyllus and H. debilis. Studies on sclerotinia resistance (Sclerotinia sclerotiorum) were carried out in field conditions and in greenhouses. Different ways of artificial inoculation were applied and the most effective was that with direct mycelium setting in different uncovered parts of the plant (Christov et al., 2004). High tolerance to Sclerotinia sclerotiorum was observed in some forms originated from Helianthus eggertii, H. pauciflorus, H. smithii, H. praecox, H. petiolaris, H. argophyllus, and H. annuus (w.f.). This tolerance referred to those pathogens forms which infect the head, stem, and the basal part of sunflower stem. Full resistance to powdery mildew (Erysiphe cichoracearum D.C.) was established in hybrid forms originated from the species H. decapetalus, H. glaucophyllus, H. giganteus, H. mollis, H. ciliaris, H. laevigatus, H. debilis, H. tuberosus, and H. resinosus. The resistance transferred from the species H. decapetalus was determined by a single dominant gene. The investigations on alternaria resistance (Alternaria helianthi (Hansf.) Tubaki and Nishihara and A. zinniae Pape) began later. A more detail study of wild species was done during the period At that time the first crosses for creating hybrid forms with resistance to alternaria were carried out. After that only hybrid forms were tested. During the last years the method of Encheva and Kiryakov (2002) was applied. Some of the obtained results were presented in Table 3. Table 3: Characterization of sunflower lines, obtained by interspecific hybridization for resistance to phomopsis, phoma, alternaria and sclerotinia Accession, pedigree Phomopsis, gr. 0-4 Resistance to, grades Phoma, gr. 0-4 Alternaria, gr. 0-4 Sclerotinia, gr. 0-5 Sc-2 L-6116B Sc-3 (c.s. H. debilis) Sc-5 (c.s. H. pauciflorus) Sc-8 (c.s. H. argophyllus) Sc-9 (c.s. H. argophyllus) Races E, F and G of the parasite broomrape (Orobanche cumana Wallr.) were spread in Bulgaria. The last two races appeared in quick succession and this aggravated sunflower breeding. During the last 20 years a sufficient number of sunflower lines resistant to race E of broomrape were developed. From 2008 on the aim of the breeding work has been to develop lines resistant to race G of the parasite.

6 42 HELIA, 35, Nr. 57, p.p , (2012) In creating forms resistant to broomrape 16 wild Helianthus species were used (H. tuberosus, H. pauciflorus, H. eggertii, H. xlaetiflorus, H. decapetalus, H. hirsutus, H. divaricatus, H. giganteus, H. maximiliani, H. nuttallii ssp. rydbergii, H. salicifolius, H. smithii, H. annuus (w.f.), H. argophyllus, H. debilis, H. petiolaris and H. praecox). Full resistance to the parasite was exhibited by some new lines such as 7019 R, 7203 R, C 23/1, C 41, C 46, C 48, C 55, C 56, etc. (Table 4). Table 4: Characterization of sunflower lines, obtained by interspecific hybridization and resistant to broomrape, harvest 2009 Accession, pedigree Resistance to Seed oil broomrape, % downy mildew, % content, % Generation PR-1/8 (c.s. H. pauciflorus) PR-9/8 (c.s. H. tuberosus) PR-19/8 (c.s. H. divaricatus) PR-25/8 (c.s H. pauciflorus) PR-35/8 (c.s H. hirsutus) PR-41/8 (c.s. H. divaricatus) PR-47/8 (c.s. H. bolanderi) New sunflower forms with high seed oil content Some accessions of wild Helianthus species could be used as sources for high seed oil content in sunflower. This conclusion is based on the results established for different hybrid forms obtained by applying interspecific hybridization. Sunflower forms and lines with high seed oil content were obtained from hybrids with participation of the species H. eggertii, H. pauciflorus (rigidus), H. smithii, H. hirsutus, H. annuus (w.f.), H. nuttallii ssp. rydbergii, H. pumilus, etc. Some of the results for seed oil content are presented in Tables 2, 4 and 6. New sources of cms The total number of the new cms sources was 15 (Table 5). Some of the sources differed from cms PET 1 significantly. Genes restoring fertility were found for all the sources. New sunflower forms with Rf genes (R lines) Thus far, more than 3,900 new R forms have been selected and obtained including 1,306 R lines which are fixed and named. All of them are resistant to downy mildew. Some of them were resistant to phomopsis and broomrape. There were lines which showed resistance to phoma and others which even had tolerance to sclerotinia. Part of these lines is presented in Table 6. New sunflower forms with normal cytoplasm (B lines) New B lines were created only from forms obtained by interspecific hybridization. The total number of B lines developed (fixed) by the year 2010 is 289. The

7 HELIA, 35, Nr. 57, p.p , (2012) 43 stem height varies from 45 to 180 cm and the vegetation period from 86 to 125 days. Thousand seed weight varies from 30 to 125 g and seed oil content from 40 to 54%. Some B lines show resistance to phomopsis and others to downy mildew and broomrape. Such lines are 6066B, 6101B, 6134B, 6149B, 6488B, 6748B, etc. Sterile analogues were developed for all the B lines in cms PET 1. Sterile analogues for the rest of the cms sources were created for four of the new lines with the aim to enable their use in some experiments such as the evaluation of the cytoplasmic effect on some agronomic characters of the new hybrids. Table 5: Sources of cms produced by interspecific hybridization Origin Obtained in generation Year of observation Year reported DAI code F.A.O. code H. annuus E F AN-67 ANN-10 H. annuus E F AN-58 ANN-11 H. annuus E F AN-2-1 ANN-12 H. annuus E F AN-2-2 ANN-13 H. argophyllus E F ARG-1 ARG-1 H. argophyllus E BC ARG-3 ARG-3 H. argophyllus E F ARG-2 ARG-2 H. debilis E F DV-10 DEB-1 H. petiolaris E BC 1 F Pet-34 PET-4 H. praecox E F PHIR-27 PRH-1 H. praecox E F PRUN-29 PRR-1 H. rigidus M BC 1 F Rig-28 RIG-2 H. strumosus M BC 1 F Strum-56 STR-1 H. argophyllus E-007 BC 1 F ARG-4 ARG-4 H. argophyllus E-006 new BC ARG-3-M-1 ARG3M1 Table 6: Characterization of R lines produced by interspecific hybridization, harvest 2010 Plant height New sunflower hybrid combinations Head diameter Vegetation period Seed oil content Generation No. Origin cm cm days % PR-1/8 c.s. H. pauciflorus M * PR-13/8 c.s. H. pumilus M * PR-41/8 c.s. H. divaricatus M * PR-47/8 c.s. H. bolanderi E * C 23/1 c.s. H. debilis E * C 55 c.s. H. debilis E * C 56 c.s. H. hirsutus M * *branched forms Two groups of combinations were made. The first combination included crosses between old, confirmed Bulgarian A (B) lines with R lines obtained from

8 44 HELIA, 35, Nr. 57, p.p , (2012) interspecific hybrids and the second group included crosses between new A (B) lines obtained using mutagenesis and R lines obtained by interspecific hybridization. There are small number of hybrid combinations created from B lines obtained from wide hybridization and R lines obtained by the same method. New sunflower hybrid varieties in registration New sunflower hybrids were developed which increased the standard in sunflower seed yield and seed oil content per unit area. Five of these - Musala, Mura, Maritsa, Mesta and Magura - were registered with the State Variety Commission at the end of The paternal forms of the hybrids Musala, Mura, Maritsa, Mesta and Magura were created from materials obtained by interspecific hybridization. REFERENCES Christov, M., and Petrov, P., New source of Rf genes for cms on the basis of H. petiolaris. Genetics and Selection 21(5). Christov, M., Study on wild species Helianthus with aim of using in sunflower breeding. Ph.D. Dissertation, Bulgarian Academy of Agriculture, Sofia. Christov, M., 1990a. A new source of cytoplasmic male sterility in sunflower originated from H. argophyllus. Helia 13: Christov, M., Shindrova, P. and Encheva, V., Phytopathological characterization of wild species in the genus Helianthus in view of their use in breeding for resistance. Genet. Breed. 25: Christov, M. 1996b. Hybridization of cultivated sunflower and wild Helianthus species. pp In: P.D.S. Caligari and D.J.N. Hind [eds.], Compositae: Biology and Utilization, Vol. 2. Proc. Intl. Composite Conf., The Royal Botanic Gardens, 24 July-5 August Kew, London. Christov, M., Shindrova, P. and Encheva, V., Transfer of new characters from wild Helianthus species to cultivated sunflower. Genet. a Slecht. 32(4): Christov, M., Production of new cms sources in sunflower. Helia 22(31): Christov, M., Kiryakov, I., Shindrova, P. and Encheva, V. and Christova, M., Evaluation of new interspecific and intergeneric sunflowerhybrids for resistance to Sclerotinia sclerotiorum. In: Proc. 16 th Int. Sunfl. Conf., Fargo, North Dakota, USA, Int. Sunfl. Assoc., Paris, France. II: Christov, M., Helianthus species in breeding research in sunflower. pp In: L. Velasco [ed.], Proc 17 th Intl. Sunflower Conf., Cordoba, Spain, 8-12 June, Intl. Sunflower Assoc., Paris, France. Encheva, V. and Kiryakov, I., A method for evalution of sunflower resistance to Diaporthe/ Phomopsis helianthi Munt.-Cvet. et al. Bulgarian Journal of Agricultural Science 8: Enns, H., Dorrell, D.G., Hoes, J.A. and Chubb, W.O., Sunflower research. Progress report. In: Proc. 4 th Int. Sunfl. Conf., Memphis, Tennessee. Int. Sunfl. Assoc., Paris, France. pp Fernandez-Martinez, J.M., Malero-Vara, J,. Munoz-Ruz, J., Ruso, J. and Domingez, J., Selection of wild and cultivated sunflower for resistance to a new broomrape race that overcomes resistance of the Or 5 gene. Crop Science 40(2): Fick, G.N., Zimmer, D.E., Dominguez-Gimenez, J. and Rehder, D.A., Fertility restoration and variability for plant and seed characteristics in wild sunflower. In: Proc. 4 th Int. Sunfl. Conf., Bucharest, Int. Sunfl. Assoc., Paris, France. Pp Hristova-Cherbadzi, M., Study of new sunflower forms by remote hybridization. Ph.D. Dissertation, Sofia. Ivanov, P., Christov, M., Ivanova, I. and Nikolova, V., Study of seed oil and protrin quality of some Bidens tripartita accessions. Helia 19(25): Jan, C.C., and Chandler, J.M., Transfer of powdery mildew resistance from Helianthus debilis Nutt. to cultivated sunflower. Crop Science 25(4):

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10 46 HELIA, 35, Nr. 57, p.p , (2012)